Sterilization and Disinfection. Role of environmental factor on microorganisms

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1 Sterilization and Disinfection. Role of environmental factor on microorganisms Lecture 5 Eli Hristozova, MD, PhD Dept. Microbiology and Immunology Medical University Plovdiv

2 Why we need knowledge about this?

3 Historical perspective Food preservation to prevent food spoilage. Drying, salting, smoking of food are used over many centuries. Low water content or low ph prevents microorganisms multiplying. Prevention of disease Joseph Lister, Ignaz Semmelweis in the 1850`s used crude chemicals for surgical asepsis and hand washing

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5 Sterilization - removal or killing of all microorganisms including spores Terminology Bacteriostatic inhibits growth of bacteria /surfaces with a bacteriostatic coating against microbes/ Bactericidal kills bacteria Decontamination-removal of contaminating pathogenic microorganisms from the articles by sterilization/disinfection Sterility total absence of viable microbes as assessed by no growth on any medium

6 Terminology Disinfection elimination of pathogenic microbes (excluding spores) on inanimate objects. Antibiotics substances, produced by one microbe that inhibits/kills another microbe Septic related to sepsis (presence of pathogenic microbes/their toxins in blood or tissues) Aseptic free of pathogenic microbes (aseptic surgical instruments) Antiseptic a chemical that inhibits infection on skin or mucus membrane, such as alcohol

7 Degrees of cleanliness 1. Sterilisation remove all microbes including their spores 2. Disinfection treatment to destroy harmful microbes, but not always their spores 3. Routine cleaning reduce numbers of microorganisms - floors, furniture generally use detergent rather than disinfectant 4. Cleaning prior to disinfection to remove interfering organic matter

8 Methods of disinfection and Physical Sunlight Heat Dry heat Red heat Flaming Incineration Hot air oven Infra red Moist heat Below 100 C At 100 C Above 100 C Vibration Radiation Non-ionizing Ionizing Particular Electromagnetic Filtration Earthenware Asbestos Sintered glass Membrane sterilization Chemical Liquid Alcohols Aldehydes Phenolic Halogens Heavy metals Surface active agents Dyes Gaseous Phormaldehyde Ethylene oxide Plasma Physical-chemical

9 Disinfection and Sterilization Heat: Vital processes are possible in certain temperature ranges: minimal, optimal and maximal Bacteria are divided in 3 temperature groups: Psychrophilic Mesophilic Thermophilic

10 Disinfection and Sterilization Psychrophilic (cryophilic) extremophile microbes capable to growth and reproduce in cold temperature ranging from -15 to 30 C, optimum from 10 to 20 C. They are found in pockets of very salty water surrounded by arctic sea ice, soils. Pathogenic bacteria like Yersinia enterocolitica and Listeria monocytogenes are psychrophilic and can multiply in refrigerators.

11 Disinfection and Sterilization Mesophilic range from 10 to 45 C with optimum C. Here are all microbes with medical importance, normal flora and some microbes from soil and water. Thermophilic microbes from thermal waters, soil, putrefactive microbes, lactic acid bacteria. They live in range: 25 to 95 C, optimum: C.

12 Disinfection and Sterilization Low temperatures hold cell metabolism, increase cytoplasm viscosity and decrease membrane permeability. ANABIOSIS = stop living processes under point of refrigeration. We can preserve vaccines, bacteria, viruses, fungi at -20 to -70 C or liquid N (-196 C) for many years.

13 Disinfection and Sterilization Above maximal temperatures ->damage the metabolism. Most pathogenic bacteria and all viruses can die over C for some minutes to 1 hour. Spores of bacilli need more time: 100 C more than 2.5 h (C. botulinum)

14 Disinfection and Sterilization Implementation of heat effect on microbes: 1. Taking and transporting of clinical samples only in proper conditions (CSF - at body temperature, urine - at 4 C). 2. Cultivating only in proper temperature - 20, 28, 37, 42 C 3. Sterilization - dental and surgical instruments, drugs, vaccines, washings, etc.

15 DRT

16 DRT (D- value)

17 Sterilization Physical methods Heat (TDT) Thermal death time = the minimum time to kill microbes at a predetermined temperature in a specified environment. The curve shows the relationship between decimal reduction times for any microbe and heating temperature

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23 Sterilization Physical methods DRY HEAT: 1. Red heat 2. Flaming 3. Incineration 4. Dry heat oven 5. Infra red

24 Sterilization Physical methods 1. Red heat by holding in Bunsen flame For: bacteriological loops, straight wires, tips of forceps and searing spatulas 2. Flaming by passing through the flame a few times the article over a Bunsen flame, but not heating it to redness For: scalpels, mouth of test tubes, flasks, glass slides and cover slips Most vegetative cells are killed, but there is no guarantee that spores would die too on such short exposure

25 Loop sterilizers Ideal for laboratories and safety cabinets where the use of gas and open flames is not permitted It uses infrared heating to produce 815 C (1500 F) Optimal temperature of 900 C (1650 F)

26 Sterilization Physical methods 3. Incineration - a method of destroying contaminated material by burning them in incinerator. For: soiled dressings, animal carcasses, pathological material and bedding, etc. Suitable only for disposable articles!

27 Incinerator

28 Sterilization Physical methods - Heat 4. Dry heat oven For: Metallic instruments (forceps, scalpels, scissors), glasswares (petridishes, pipettes, flasks, all-glass syringes), swabs, oils, grease, petroleum jelly, pharmaceutical products. Sterilization process: perfectly dry before placing them inside to avoid breakage. Articles must be placed at sufficient distance -> free circulation of air in between. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton wool. Individual glass articles must be wrapped in craft paper or aluminum foils. Sterilization cycles: 1.5 to 2 hours at 160 C OR 1 hour at 180 C. Increasing temperature by 10 degrees shortens the sterilizing time by 50%!

29 Dry heat oven

30 Sterilization Physical Dry heat oven methods - Heat

31 Sterilization Physical methods - Heat Sterilization control of dry heat oven: Physical: Temperature chart recorder and thermocouple. Chemical: Browne s tube No.3 (green spot, color changes from red to green) Biological: 106 spores of Bacillus subtilis var. niger or Clostridium tetani on paper strips are placed inside envelopes and then placed inside the hot air oven. The strips are removed and inoculated into nutrient media at 37 C for 3-5 days. Proper sterilization results in no growth. Advantages: For heat stable articles. The articles remain dry after sterilization. The only method of sterilizing oils and powders. Disadvantages: Hot air has poor penetration Takes longer time compared to autoclave

32 5. Infra red Sterilization Physical By generation of heat For: metallic instruments and glassware. methods - Heat Articles are placed in a moving conveyer belt and passed through a tunnel that is heated by infrared radiators to a temperature of 180 C. The articles are exposed to that temperature for a period of 7.5 minutes.

33 Sterilization Physical MOIST HEAT: methods - Heat 1. Below 100 C 2. At 100 C 3. Above 100 C Moist heat acts by coagulation and denaturation of proteins.

34 Vaccine bath: an apparatus, used in vaccine production; a water baths, with a temperature of 60 C for 1 hour. Sterilization Physical methods - Heat 1. Below 100 C: Pasteurization: Employed by Louis Pasteur for food and dairy industry. 2 methods of pasteurization: holder method (heated at 63 C for 30 minutes) flash method (heated at 72 C for 13 seconds; both methods followed by quickly cooling to 4 C. Ultra-High Temperature (UHT) or ultrapasteurization C for 15 sec and 149 C for 0.5 sec. suitable to destroy most milk-borne pathogens like Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, but Coxiella (causing Q-fever) may survive pasteurization.

35 Sterilization Physical methods - Heat Serum bath The contaminating bacteria in a serum preparation can be inactivated by heating in a water bath at 56 C for 1 hour on several successive days. Only vegetative bacteria are killed and spores survive. Inspissation=thickening by dehydration Method to solidify as well as disinfect egg- and serum containing media(lowenstein-jensen, Loeffler`s serum). The media are placed in the slopes of an inspissator and heated at C for 30 minutes on 3 successive days.

36 Inspissator

37 Sterilization Physical methods - Heat At a temperature 100 C Boiling: Boiling water (100 C) kills most vegetative bacteria and viruses immediately. But.certain bacterial toxins -> staphylococcal enterotoxin are heat resistant. Some bacterial spores are resistant to boiling and survive! The killing activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not required, certain metal articles and glasswares can be disinfected for minutes.

38 Sterilization Physical methods - Heat Steam at 100 C Traditionally Arnold s and Koch s steamers were used. An autoclave (with discharge tap open) can also serve the same purpose. A steamer is a metal cabinet with perforated trays to hold the articles and a conical lid. The bottom of steamer is filled with water and heated. The articles are exposed for 90 minutes. Different media - TCBS, DCA and Selenite broth are sterilized by steaming. Tyndallisation (after John Tyndall) or fractional sterilization. The vegetative bacteria are killed in the first exposure at 100C and the spores that germinate by next 2 days are killed during subsequent heating. For: gelatin and sugar containing media.

39 Above 100 C: Sterilization Physical methods - Heat Autoclave: Sterilization can be effectively achieved at a temperature above 100 C using an autoclave. In an autoclave the water is boiled in a closed chamber. As the pressure rises, the boiling point of water also rises. At a pressure of 1 Atm inside the autoclave, the temperature is said to be 121 C. Exposure of articles to this temperature for 15 minutes sterilizes them.

40 Sterilization Physical methods - Heat Advantages of steam: more penetrative power than dry air moistens the spores (moisture is essential for coagulation of proteins) Different types of autoclave: Simple pressure-cooker type laboratory autoclave-1 Atm=121 C High pressure pre-vacuum autoclave- 2 Atm=134 C

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42 Sterilization Physical methods - Heat

43 Autoclave - principle

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45 Autovclaves

46 Autoclave scheme

47 Sterilization Physical methods - Heat Articles sterilized: Culture media, dressings, certain equipment, linen, etc. Precautions: Articles should not be tightly packed, the autoclave must not be overloaded, caps of bottles and flasks should not be tight, bottles must not be overfilled. Advantage: Very effective way of sterilization, quicker than hot air oven. Disadvantages: Drenching and wetting of articles may occur, trapped air may reduce the efficacy, takes long time to cool.

48 Sterilization control: Physical temperature+ pressure records to ensure correct temperature and pressure achieved by chamber as a whole. Chemical autoclave tape changes colour colour changes if correct temperature in centre of load. Biological method includes a paper strip containing 106 spores of Geobacillus stearothermophilus. Sterilization Physical methods Heat

49 Autoclave <-> Hot air oven

50 Sterilization Physical methods Irradiation IRRADIATION 2 types of radiation are used: Non-ionizing rays are low energy rays with poor penetrative power Ionizing rays are high-energy rays with good penetrative power. Since radiation does not generate heat, it is termed "cold sterilization". In some parts of Europe, fruits and vegetables are irradiated to increase their shelf life up to 500%.

51 Sterilization Physical methods Radiation Products sterilised by radiation Tissue grafts Pharmaceutical Medical & lab Household products supplies items Cartilage Ampicillin Disposable lab Toothbrushes plastics Tendon Tetracycline Culture media Cosmetics Skin Atropine Syringes Toilet paper Heart valve Vaccines Surgical equipment Personal hygiene products Ointments Suture material

52 Sterilization Physical methods Irradiation Non-ionizing rays Microbicidal wavelength of UV rays lie in the range of nm, with 260 nm being most effective. UV rays are generated by mercury vapor lamp with germicidal effect. UV rays induce formation of thymine-thymine dimers, which inhibits DNA replication. UV readily induces mutations in cells irradiated with a non-lethal dose. Microorganisms such as bacteria-vre, MRSA, ESBL, A. baumanii, C. difficillae; viruses-influenza virus A, yeasts, etc. are inactivated within seconds.

53 Sterilization Physical methods Irradiation UV rays don t kill spores - to be of use in surface disinfection. UV are in use for hospital wards, operation theatres, virus laboratories, corridors, etc. Disadvantages: low penetrative power some bacteria have DNA repair enzymes that can overcome damage organic matter and dust prevents its reach rays are harmful to skin and eyes. It doesn't penetrate glass, paper or plastic.

54 Sterilization Biological safety cabinet with UV source

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56 Disadvantage includes poor penetrative power and requirement of sophisticated equipment. Sterilization - Physical methods Radiation Ionizing rays: Ionizing rays are of 2 types: particulate and electromagnetic rays. Electron beams are particulate in nature while gamma rays are electromagnetic in nature. Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals. Sterilization is accomplished in few seconds.

57 Sterilization - Physical methods Radiation lectromagnetic rays such as gamma rays manate from nuclear disintegration of isotopes Co60, Cs137) more penetrative power than electron beam but require longer time of exposure damage the nucleic acid of the microorganism 2.5 megarads kills all bacteria, fungi, viruses and spores disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics isadvantages: glasswares tend to become brownish impairment the flavour of certain foods by gamma rays. Bacillus pumilus E601 is used to evaluate sterilization process.

58 Sterilization - Physical methods Filtration FILTRATION Does not kill microbes, separates them out. Membrane filters with pore sizes between μm are commonly used to remove particles from solutions that can't be autoclaved. remove microbes from heat labile liquids - serum, antibiotic solutions, carbohydrates solutions, urea solution. Filtration is aided by using either (+) or (-) pressure using vacuum pumps. The older filters made of earthenware or asbestos are called depth filters.

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60 Sterilization - Physical methods Filtration 1. Earthenware (Candle) filters: diatomaceous earth or porcelain.

61 Asbestos filters

62 Sintered glass filter

63 Sterilization - Physical methods Filtration 4. Membrane filters: polymeric materials /cellulose nitrate, cellulose diacetate, polycarbonate and polyester/. Gradocol membranes -> pore diameter 0.4 μm. These filters are sterilized by autoclaving. Disadvantages: migration of filter material into the filtrate, absorption or retention of certain volume of liquid by the filters; viruses and mycoplasma could pass through. Advantages: known porosity reusable after autoclaving compatible with many chemicals.

64 Sterilization - Physical methods Filtration 5. Syringe filters - single-use filter cartridge attached to the end of a syringe for use In scientific applications, the most common sizes available are 0.2 or 0.22 µm and 0.45 µm pores. These sizes are sufficient for High-performance liquid chromatography (HPLC) use. The smallest one is with d=0.02 µm of the pores. Membrane diameters of 10 mm, 13 mm, 25 mm are common as well

65 Sterilization - Physical methods Filtration 6. Air filters Air can be filtered using HEPA (High Efficiency Particle Air) filters usually used in biological safety cabinets. HEPA filters are at least 99.97% efficient for removing particles >0.3 μm in diameter. Examples: rooms housing severely neutropenic patients operating rooms for orthopedic implant procedures.

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69 Sterilization - Physical methods SONIC AND ULTRASONIC VIBRATIONS: Sound waves of frequency >20,000 cycle/second kills bacteria and some viruses on exposing for 1 hour. The killing effect of microwaves are largely due to the heat that they generate. High frequency sound waves disrupt cells. They are used to clean and disinfect instruments as well as to reduce microbial load. This method is not reliable since many viruses and phages are not affected by these waves.

70 CHEMICAL METHODS OF DISINFECTION: Disinfectants: chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemosterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics.

71 CHEMICAL METHODS OF DISINFECTION: An ideal antiseptic or disinfectant should have following properties: wide spectrum of activity able to destroy microbes within practical period of time active in the presence of organic matter make effective contact and be wettable be active in any ph be stable have long shelf life be speedy high penetrating power non-toxic, non-allergenic, non-irritative or non-corrosive not have bad odour should not be lost on reasonable dilution not be expensive and must be available easily

72 CHEMICAL METHODS OF DISINFECTION But such an ideal disinfectant is not yet available. The level of disinfection achieved depends on: contact time temperature type and concentration of the active ingredient the presence of organic matter the type and quantum of microbial load. The chemical disinfectants at working concentrations rapidly lose their strength on standing.

73 CHEMICAL METHODS OF DISINFECTION Classification of disinfectants: 1. Based on consistency a. Liquid (Alcohols, Phenols) b. Gaseous (Formaldehyde vapor, Ethylene oxide) 2. Based on spectrum of activity a. High level b. Intermediate level c. Low level 3. Based on mechanism of action a. Action on membrane (Alcohol, detergents) b. Denaturation of cellular proteins (Alcohol, Phenol) c. Oxidation of essential sulphydryl groups of enzymes ( H2O2, Halogens) d. Alkylation of amino-, carboxyl- and hydroxyl group (Ethylene Oxide, Formaldehyde) e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

74 CHEMICAL METHODS OF DISINFECTION ALCOHOLS Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein. Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols-96%. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces and clinical thermometers. Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods. Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

75 CHEMICAL METHODS OF DISINFECTION ALDEHYDES: Mode of action: Alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic acids. It kills all microorganisms, including spores. Examples: Formaldehyde, Gluteraldehyde Application: 40% Formaldehyde (formalin) - for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms,etc. Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. 2% formaldehyde at 40 C for 20 minutes is used to disinfect wool and 0.25% at 60 C for 6 hours to disinfect animal hair and bristles. Disadvantages: Vapors can cuse irritation, has poor penetration; activity is reduced in the presence of protein.

76 CHEMICAL METHODS OF DISINFECTION PHENOLS: Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes. Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol) Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. The corrosive phenolics are used for disinfection of ward floors, in discarding jars in laboratories and disinfection of bedpans.

77 CHEMICAL METHODS OF DISINFECTION Chlorhexidine - for skin disinfection, or as an aqueous solution for wound irrigation, for antiseptic hand wash. 20% Chlorhexidine gluconate solution- for pre-operative hand, skin preparation, for general skin disinfection. Chlorhexidine gluconate - mixed with quaternary ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon). Chloroxylenols are less irritant and can be used for topical purposes and are more effective against Gram(+) bacteria than Gram(-) bacteria. Hexachlorophene - effect over Gram(+) bacteria but poor effect over Gram(-) bacteria, mycobacteria, fungi and viruses. Triclosan - good activity against Gram(+) bacteria and effective to some extent against many Gram(-) bacteria including Pseudomonas, a fair activity on fungi and viruses. Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps. Chloroxylenol is inactivated by hard water

78 CHEMICAL METHODS OF DISINFECTION

79 CHEMICAL METHODS OF DISINFECTION HALOGENS: Mode of action: Oxidizing agents -> oxidation of sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid = microbicidal. Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores) Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. 10% Povidone Iodine is used undiluted in preand postoperative skin disinfection. Chlorine gas is used to bleach water.

80 CHEMICAL METHODS OF DISINFECTION Household bleach: Disinfect floors in a stock dilution of 1:10 In higher concentrations -> swimming pools 0.5% sodium hypochlorite - in serology and virology Used at a dilution of 1:10 in decontamination of spillage of infectious material. Mercuric chloride is used as a disinfectant. Disadvantages: Rapidly inactivated in the presence of organic matter. Iodine - corrosive and staining. Bleach solution corrosive; corrode stainless steel surface

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82 CHEMICAL METHODS OF DISINFECTION HEAVY METALS: Mode of action: precipitation of proteins and oxidation of sulfydryl groups; bacteriostatic. Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g., mercurochrome, merthiolate) Applications: 1% silver nitrate solution -> treatment of opthalmia neonatorum. Silver sulphadiazine - topically prevent colonization and infection of burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in preservation of serum. Copper salts are used as a fungicide. Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

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84 CHEMICAL METHODS OF DISINFECTION SURFACE ACTIVE AGENTS: Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. They disrupt membrane resulting in leakage of cell constituents. Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents containing negatively charged long chain hydrocarbon are called anionic detergents. These include soaps and bile salts. Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride are cationic detergents. Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals. Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter.

85 CHEMICAL METHODS OF DISINFECTION DYES Mode of action: bactericidal because of their interaction with bacterial nucleic acids. Examples: ANILINE DYES - crystal violet, malachite green and brilliant green Applications: topically as antiseptics to treat mild burns as paint on the skin to treat bacterial skin infections The dyes are used as selective agents in certain selective media.

86 CHEMICAL METHODS OF DISINFECTION HYDROGEN PEROXIDE: Mode of action: release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA. Application: at 6% concentration-decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution - for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal. Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically reduces its activity.

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88 CHEMICAL METHODS OF DISINFECTION ETHYLENE OXIDE (EO) Mode of action: An alkylating agent. Properties: With sweet ethereal odor, flammable. Application: It is a highly effective chemisterilant, kills spores rapidly; good penetration. Sterilize heat labile articles - bedding, textiles, rubber, plastics, syringes, disposable petri dishes, heart-lung machine, respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var. niger. Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and carcinogenic

89 The role of environmental factors on microorganisms Microbes are in the connection with the factors of the environment: chemical physical biological Knowledge of their impact have a great importance for: Optimal conditions for cultivating Fast diagnosis of infection Eradication of microbes from site of infection

90 The role of environmental factors on microorganisms Physical factors Heat microbes have optimal temperature for their growth 3 main groups: Phychrophiles t C between 10-30C Yersinia pestis, Y. enetrocolitica Mesophyles - t C between 20-40C, microbes with medical importance E. coli Termophyles t C between 25-95C, thermal springs microbes, etc.

91 The role of environmental factors on microorganisms Physical factors Drying Microbes have different sensitivity towards water loss: Meningococcus, Haemophillus, Treponaema pallida (syphilis) are very sensitive Spores of bacilli are very resistant (Bacillus anthracis, Clostridium)

92 The role of environmental factors on microorganisms Physical factors ph of the medium Optimal ph Weakly acid (6.4-7) Mycobacterium tuberculosis Alkaline ph ( ) Vibrio cholerae Implementation: Changes of ph have impact on regulation of fermentations, antibiotic production, beverages, etc.

93 The role of environmental factors on microorganisms Physical factors osmotic pressure Intracellular osmotic pressure of microbes is from Pa to Pa, measured on artificial nutrient media at 0.5% NaCl But some microbes in oceans (halophiles) can survive at greater osmotic pressure

94 The role of environmental factors on microorganisms Physical factors irradiation UV Rays - affect DNA of microbes, cause a mutagenic or lethal effect. Bactericidal lamps ( nm) sterilize the air in operation halls, dental medicine cabinets, paediatric units, etc. Ionising irradiation (β-particles and electrons) Electromagnet irradiation (Roentgen and ϒ-rays) with bigger energy compared to the UV rays

95 Physical factors Oligodynamic The role of environmental factors on microorganisms The ability of some metals (Au, Ag) to kill bacteria trough releasing of metal ions onto the surface of microbial cell. The metabolite processes are interrupted Implementation dental medicine Au, Ag, Colargol (Argentum colloidalae pulvis)

96 The role of environmental factors on microorganisms hysical factors Sound nergy Ultrasound over Hz/s leads to cavitation >bubbles of solubilised gases that attack the cell and lead to its destruction Implementation: Dental medicine practice, for extraction of enzymes, endotoxins, antigens, etc.

97 The role of environmental factors on microorganisms Chemical factors Disinfection and sterilization Bactericidal only prevent bacterial growth Bacteriostatic stops live processes in the cell

98 The role of environmental factors on microorganisms Biological factors Forms of coexistence: Symbiosis - aerobe and anaerobe microbes in gasgangrenae, the satellite phenomenon between S. aureus and H. influenzae Antagonism between normal flora in human body and pathogenic microbes Antibiotics a products of antibiosis a process of suppression of a growth from substances released from other microbes (actynomycetes, moulds, bacteria) Bacteriophage

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